How HIV-1 Nef hijacks the AP-2 clathrin adaptor to downregulate CD4.

5 MB·English

by  Ren, Xuefeng

RESEARCH ARTICLE
elife.elifesciences.org
How HIV-1 Nef hijacks the AP-2 clathrin
adaptor to downregulate CD4
Xuefeng Ren1,2,3, Sang Yoon Park3, Juan S Bonifacino3*, James H Hurley1,2*
1Department of Molecular and Cell Biology, University of California, Berkeley,
Berkeley, United States; 2California Institute for Quantitative Biosciences, University
of California, Berkeley, Berkeley, United States; 3Cell Biology and Metabolism
Program, Eunice Kennedy Shriver National Institute of Child Health and Human
Development, National Institutes of Health, Bethesda, United States
Abstract The Nef protein of HIV-1 downregulates the cell surface co-receptor CD4 by hijacking
the clathrin adaptor complex AP-2. The structural basis for the hijacking of AP-2 by Nef is revealed
by a 2.9 Å crystal structure of Nef bound to the α and σ2 subunits of AP-2. Nef binds to AP-2 via its
central loop (residues 149–179) and its core. The determinants for Nef binding include residues that
directly contact AP-2 and others that stabilize the binding-competent conformation of the central
loop. Residues involved in both direct and indirect interactions are required for the binding of Nef
to AP-2 and for downregulation of CD4. These results lead to a model for the docking of the full
AP-2 tetramer to membranes as bound to Nef, such that the cytosolic tail of CD4 is situated to
interact with its binding site on Nef.
DOI: 10.7554/eLife.01754.001
Introduction
The human immunodeficiency virus type 1 (HIV-1) is a lentivirus that causes acquired immunodefi-
*For correspondence:
[email protected] (JSB); ciency syndrome (AIDS). HIV-1 has a small genome encoding the main structural proteins Gag, Pol and
[email protected] (JHH) Env, the regulatory proteins Tat and Rev, and the accessory proteins Nef, Vif, Vpr, and Vpu (Frankel
and Young, 1998). During viral maturation, Pol is proteolytically cleaved to generate three proteins with
Competing interests: The
enzymatic activity: the viral protease, integrase, and reverse transcriptase. These enzymes are the main
authors declare that no
targets for chemotherapeutic agents currently in use for the prevention and treatment of AIDS.
competing interests exist.
Combination therapies with these agents have dramatically reduced HIV-1 transmission as well as HIV-1-
Funding: See page 16 associated morbidity and mortality. However, concerns about the development of drug resistance in
Received: 21 October 2013 addition to their side effects have fueled a continued search for additional targets. The accessory
Accepted: 09 December 2013 protein Nef was recognized early on as a potential target for inhibition of the pathogenic effects of
Published: 28 January 2014 HIV-1 (Coleman et al., 2001; Foster and Garcia, 2008). Although not essential for infection in cell
culture, Nef enhances viral replication and disease progression in vivo. The pathogenic effects of
Reviewing editor: Wesley
Nef are underscored by the observation that patients infected with Nef-deficient strains of HIV-1
Sundquist, University of Utah,
often do not develop AIDS for over 10 years even if untreated (these patients are referred to as ‘long-
United States
term non-progressors’ or ‘slow progressors’) (Deacon et al., 1995; Kirchhoff et al., 1995; Gorry
This is an open-access article, et al., 2007). Inhibition of Nef thus holds the promise to have a similarly beneficial effect. To date,
free of all copyright, and may be
however, this potential has not been realized mainly because Nef has no enzymatic activity and its
freely reproduced, distributed,
mechanisms of action are insufficiently understood.
transmitted, modified, built
At the cellular level, Nef has been ascribed multiple functions, of which the best characterized and
upon, or otherwise used by
most critical for pathogenesis is the downregulation of CD4 from the surface of infected cells (Guy
anyone for any lawful purpose.
The work is made available under et al., 1987; Garcia and Miller, 1991; Carl et al., 2000; Glushakova et al., 2001). CD4 is a transmem-
the Creative Commons CC0 brane protein that acts as a co-receptor in both the host’s immune response and the initial binding of
public domain dedication. HIV-1 to their target cells (Bowers et al., 1997). Nef-induced CD4 downregulation interferes with the
Ren et al. eLife 2014;3:e01754. DOI: 10.7554/eLife.01754 1 of 19
Research article Biophysics and structural biology
eLife digest Infection by a pathogen, such as a bacterium or virus, activates both the innate
immune response—which is immediate but not specific to the pathogen—and the adaptive immune
response, which is stronger and specific to the pathogen. White blood cells called CD4+ T helper
cells play an important role in the early stages of the adaptive immune response by helping to
activate and regulate other white blood cells that go on to eradicate the pathogen.
HIV-1 is a retrovirus that infects immune cells that have the CD4 receptor on their surface,
including CD4+ T helper cells. As the number of worker CD4+ T helper cells falls, the adaptive
immune response gradually weakens, and the HIV-1 infected individual becomes increasingly
susceptible to infection and disease. An individual is said to develop AIDS when either their CD4+ T
helper cell count falls below 200 cells per microliter or they begin to experience specific diseases
associated with the HIV-1 infection.
In an effort to prevent and treat AIDS, researchers have worked to understand the HIV-1 genome
and have developed medicines that target the enzymatic activity of viral proteins involved in viral
replication. When used in combination, these drugs have helped to reduce transmission of HIV-1,
and also to reduce deaths from the disease. However, worries about side effects and drug
resistance mean that there is a need to develop new drugs.
The HIV-1 genome codes for a number of accessory proteins, including a protein known as Nef
that attacks the CD4+ T helper cells, removing the CD4 protein that gives the cells their name. This
reduces the ability of the T cells to activate the immune system and allows the virus to spread. Nef
acts by forming a complex with a protein called AP-2 in the T cells, and this complex then interacts
with the CD4 proteins, causing them to be internalized and then destroyed inside the cells.
Ren et al. have now worked out the structure of the Nef:AP-2 complex at the molecular level and
identified the amino acid residues within the Nef protein that interact with the AP-2 protein. This
allowed Ren et al. to propose a detailed model of the interaction between the complex and the
CD4 protein, and how this leads to the protein being destroyed. This information could be used to
develop drugs that work by blocking the amino residues on AP-2 that bind to Nef. Moreover, since
these sites are not susceptible to rapid mutations, such drugs are less likely to encounter the
problem of drug resistance.
DOI: 10.7554/eLife.01754.002
immune system (Skowronski et al., 1993), prevents superinfection (Benson et al., 1993) and
promotes virion release (Lama et al., 1999; Ross et al., 1999), all of which contribute to enhanced
HIV-1 propagation. HIV-1 Nef is a small, polymorphic protein of 200–215 amino acids having a myris-
toylated N-terminus. X-ray crystallography (Lee et al., 1996; Arold et al., 1997; Horenkamp et al.,
2011; Jia et al., 2012) and NMR (Grzesiek et al., 1996a, 1997) have shown that Nef has a folded core
(residues 55–65 and 84–203), with flexible N-terminal (residues 1–54) and C-terminal (residues
204–206) segments, and a central flexible loop (residues 149–179) (residue numbers correspond to
the NL4-3 strain of HIV-1). CD4 downregulation depends on both Nef myristoylation (Aiken et al.,
1994) and specific residues in the loop, including Leu164 and Leu165 (Bresnahan et al., 1998; Craig
et al., 1998; Greenberg et al., 1998; Janvier et al., 2003), which are in a sequence context fitting the
[DE]XXXL[LI] motif for dileucine-based sorting signals (Bonifacino and Traub, 2003), and the diacidic
motif, Asp174-Asp175 (Aiken et al., 1996; Lindwasser et al., 2008). Myristoylation allows recruitment
of Nef from the cytosol to the inner leaflet of the plasma membrane (Yu and Felsted, 1992) while the
loop engages the clathrin-associated adaptor protein 2 (AP-2) complex (Jin et al., 2005; Chaudhuri
et al., 2007; Doray et al., 2007; Lindwasser et al., 2008; Mattera et al., 2011; Jin et al., 2013). The
Nef:AP-2 complex interacts with the cytosolic tail of CD4, leading to the cooperative assembly of a
tripartite Nef:AP-2:CD4 complex (Chaudhuri et al., 2009). This complex nucleates the formation of
clathrin-coated pits (Foti et al., 1997; Greenberg et al., 1997; Burtey et al., 2007) that mediate
rapid internalization of CD4, followed by its delivery to lysosomes via the multivesicular body pathway
(Aiken et al., 1994; Rhee and Marsh, 1994; daSilva et al., 2009).
Biochemical analyses have demonstrated that binding of Nef to AP-2 is direct and dependent on
the dileucine and diacidic motifs, and other residues, in the Nef loop (Chaudhuri et al., 2007; Doray
et al., 2007; Lindwasser et al., 2008; Chaudhuri et al., 2009; Mattera et al., 2011). AP-2 is a
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Research article Biophysics and structural biology
heterotetramer composed of α, β2, μ2 and σ2 subunits. The N-terminal ‘trunk’ domains of α and β2
together with the whole μ2 and σ2 subunits constitute the core of the complex, whereas the C-terminal
‘hinge’ and ‘ear’ domains of α and β2 form long projections that extend from the core (Owen et al.,
2004). The AP-2 core undergoes a large conformation change from a ‘locked’ to an ‘open’ conformation
that allows it to bind sorting signals and to be recruited to membranes via interaction with the
phosphatidylinositol lipid PI(4,5)P (Jackson et al., 2010). Nef has been shown to bind to the α–σ2
2
‘hemicomplex’ (Chaudhuri et al., 2007; Doray et al., 2007). The σ2 subunit (with a small contribution
from the α subunit), harbors a binding site for [DE]XXXL[LI]-type signals from host cell proteins (Kelly
et al., 2008; Jackson et al., 2010; Mattera et al., 2011). Mutational analyses have shown that
this site is also required for Nef binding, most likely through recognition of the Nef dileucine motif
(Mattera et al., 2011). The α subunit has an additional site, comprising Lys298 and Arg341, which is also
required for Nef binding and CD4 downregulation (Chaudhuri et al., 2009). Although it is tempting
to hypothesize that these basic residues interact with the Nef diacidic motif, there is currently no
direct evidence for such an interaction. Importantly, this second site on α is not known to participate
in any host cell function, making it a possible target for selective interference.
Despite progress in the identification of determinants of the Nef-AP-2 interaction, the conformation
of the Nef loop when bound to AP-2 and the molecular details of the interaction are not known. To
elucidate the structural basis for this interaction, we have solved the crystal structure at 2.9 Å resolution
of Nef (residues 54–203) in complex with the α (residues 1–396) and σ2 (full-length) subunits of AP-2.
The structure reveals that the entire central loop is well ordered, and that most of it contacts the
α−σ2 hemicomplex. The Nef core is directly involved in contacts as well as serves as a scaffold to
position the central loop. The structure leads to a model for the docking of HIV-1 Nef onto the plasma
membrane in conjunction with AP-2, and suggests how the AP-2:Nef complex binds to the CD4
cytosolic tail in the membrane setting.
Results
High-affinity binding of HIV-1 Nef to the α–σ2 hemicomplex
In the absence of PI(4,5)P-containing membranes, the AP-2 core is in a locked conformation that has
2
low affinity for both physiological cargoes and Nef. Previously, we assayed a version of the AP-2 core
in which the μ2 C-terminal domain was deleted so as to destabilize the locked conformation. This
construct bound to HIV-1 Nef with K = 6 μM as judged by surface plasmon resonance (Chaudhuri
d
et al., 2007). However, the conformational lability introduced into this construct made it unsuitable
for crystallization. We built on the finding that Nef interacts with the α–σ2 hemicomplex as judged by
yeast three hybrid (Y3H) (Chaudhuri et al., 2007) and pulldown assays (Doray et al., 2007).
Hemicomplex constructs including the full α trunk domain are poorly stable, because a large amount of
hydrophobic surface area is exposed on the C-terminal part of the trunk domain when the hemicomplex
is excised from the intact AP-2 core. A truncated version of the homologous γ-ζ hemicomplex of
COPI including the first 17 helices of the γ trunk domain was found to be suitable for crystallography
(Yu et al., 2012). We designed a construct comprising the first 19 helices (residues 1–396) of the
α trunk domain and co-expressed it with full-length σ2 (Figure 1A,B). This portion of the α trunk
includes all of the Nef-interacting residues of α that have been documented to date. This construct
bound to the HIV-1 NL4-3 Nef (54–203) (hereafter, ‘Nef’) with K = 1.8 μM and 1:1 stoichiometry,
d
as determined by isothermal titration calorimetry (ITC) (Figure 1C). The comparatively high affinity
of the interaction and the congruence with previous results with the tetrameric construct led us to
conclude that this hemicomplex included all the major determinants of the AP-2:Nef interaction.
Crystal structure of the α–σ2:Nef complex
Nef was co-crystallized with the α1–396 form of the AP-2 α–σ2 hemicomplex (hereafter, ‘α–σ2’). The
structure was determined by molecular replacement at 2.9 Å resolution (Figure 2A,B; Table 1). The
asymmetric unit contains four Nef:α–σ2 complexes, all in similar conformations, with small variations in
the quality of the electron density. The description will focus on the B, C, and D chains, for which the
Nef:α–σ2 interface is most clearly visualized. Nef buries 1170 Å2 in this interface, of which two-thirds
is buried against σ2 and the remainder against α. The α–σ2 unit is essentially rigid, in a conformation
identical to that seen in other structures of the AP-2 complex (Collins et al., 2002; Kelly et al., 2008;
Jackson et al., 2010). The Nef core (excluding the central loop 149–179) contains the five α-helices
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Figure 1. Nef binds with low micromolar affinity to the AP-2 α–σ2 hemicomplex. (A) Schematic representation of
AP-2 α–σ2 and Nef protein constructs. AP-2 α (1–396) (cyan) and full-length σ2 (magenta) were generated as a
stable subcomplex and the interaction with the indicated Nef construct (54–203) (orange) was analyzed. (B) SDS gel
of recombinant AP-2 α–σ2 and Nef proteins. (C) Isothermal titration calorimetry of the titration of His-tagged Nef
(54–203) to the AP-2 α–σ2 hemicomplex. The upper panel shows the differential heat released when Nef (0.6 mM)
was injected into AP-2 α–σ2 solution (40 μM) in 2.1 μl aliquots.
DOI: 10.7554/eLife.01754.003
(H2, H3, H6, H7, H8) and five-stranded β-sheet visualized in other Nef crystal structures (Lee et al.,
1996; Figure 2C). The core also manifests a poorly ordered N-terminal helix (H1) spanning residues
55–65, which was not visualized in all of the chains. The identity of this helix was provisionally assigned
on the basis of the only other crystal structure in which this region was visualized (Breuer et al., 2011).
This helix was first identified by solution NMR and contains the primary binding site for CD4 (Grzesiek
et al., 1996a). In contrast to most other crystal structures, the central loop from residues 149–179
was visualized in its entirety (Figure 2A). The central loop contains two additional helices, one from
residues 150–157 (H4), and the other a single turn from 167–170 (H5). The central loop interacts
extensively both with the α and σ2 subunits (Figure 2D), with the greatest contact surface involving
σ2. The core interacts primarily via a network of interactions between helix H3 and the α subunit.
The landmarks within the central loop are helix H4 (150–157), the acidic-dileucine motif
(160–165), helix H5 (167–170), and finally a series of turns centered on Met173 (171–179) (Figure 2D).
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Figure 2. Crystal structure of the AP-2 α–σ2:Nef complex. (A) F-F omit map of Nef loop (149–179) with the
0 c
final model superimposed. The map is contoured at 2.0 σ. (B) Overall ribbon representation of AP-2 α (cyan)
and AP-2 σ2 subunits (magenta) in complex with Nef (orange). (C) Detailed ribbon model of Nef (orange)
with the secondary structures indicated. (D) Ribbon model of the Nef central loop (149–179), which includes
helix H4 (150–157), the acidic-dileucine motif ( ExxxLL ), helix H5 (167–170), and the C-terminal turn-rich
160 165
segment (171–179).
DOI: 10.7554/eLife.01754.004
The intramolecular interactions of H4 are with other sections of the central loop, explaining why this
helix has not been observed in other structures of Nef. The Leu164-Leu165 pair of the dileucine motif
anchors the loop in a pocket on σ2 just as seen for a dileucine peptide bound to the unlatched AP-2
tetramer (Kelly et al., 2008; Figure 3A). The dileucine peptide in the unlatched structure (Kelly et al.,
2008) used for comparison is derived from CD4, but it is important to emphasize that this binding
mode is dependent on phosphorylation and is unrelated to Nef-dependent downregulation. The pocket
walls are formed by hydrophobic residues of σ2. Nef Glu160 of the motif binds to basic residues on
both σ2 and α (Figure 3B). Residues of H4, notably Glu154, make electrostatic interactions with a
second basic patch on σ2 (Figure 3C). H4 and the dileucine motif have little or no interaction with the
Nef core, and their conformation seems to be specified by their interactions with α–σ2.
In contrast, helix H5 and the C-terminal turn segment are sandwiched between α–σ2 and the Nef core.
Their structure clearly depends on the interactions with α–σ2, since they are not otherwise visualized
in this conformation. However, the Nef core also seems to have an important role in organizing this
segment. H5 packs against the β-sheet of the core, and serves primarily to orient the hydrophobic
loop with respect to the dileucine motif. The tight turns of the C-terminal part of the loop serve to
project a number of charged and hydrophobic side chains into complementary interactions with both
α and σ2 (Figure 3D). This unusual sequence of turns is anchored at its ends by H5 and by the strand
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Table 1. Statistics of crystallographic data collection and refinement
Construct AP-2: σ2 (1–143), α (1–396); Nef (54–203)
Data collection
X-ray source APS 22-ID
Wavelength (Å) 1.0000
Space group P222
1 1 1
Cell dimensions a = 109.56 Å, b = 168.03 Å, c = 200.20 Å, α = β = γ = 90°
Resolution (Å) (last shell) 50.00–2.90 (3.00–2.90)
Unique reflections 80,188
R * (%) 18.4 (53.9)
sym
I/σ 7.4 (1.9)
Completeness 95.8 (80.2)
Redundancy 5.2 (3.2)
Refinement
R /R (%) 21.9/26.7
work free
Average B values (Å2) 39.5
Number of protein atoms 21,588
R.m.s. bond length deviation (Å) 0.015
R.m.s. bond angle deviation (°) 1.16
Ramachandran Plot (%)
Favored 98.4
allowed 1.2
outlier 0.4
*R = ΣΣ|I(h)−<I>|/ΣΣI(h), where I is the observed intensity and <I> is the average intensity of multiple
sym h i i h ii
observations of symmetry-related reflections.
DOI: 10.7554/eLife.01754.005
β5 of the core. The whole turn-rich section of the loop from 171–179 is anchored internally by a
hydrogen bond between Nef Asp174 and the main-chain amide of Gln104, and by a partially buried
salt bridge between Nef Asp175 of the loop and Nef Arg134 of the core β sheet (Figure 3E).
The structure also reveals that residues of Nef helix H3 of the core directly contact AP-2. In particular,
Gln104, Arg105, and Asp108 bind to a basic patch on α (Figure 3F). This polar interface adjoins the
mixed polar and hydrophobic interface created by the C-terminal turn segment of the central loop.
There is one other minor interaction with the core region, involving Nef Pro129 of the β2-β3 loop.
The Pro side-chain forms van der Waals interactions with atoms of the α Arg341 side-chain. While the
central loop clearly dominates the interactions overall, the Nef core interactions are also significant,
and represents one of the completely unexpected findings from the structural analysis.
Nef residues in AP-2 binding and CD4 downregulation
The Nef-AP-2 interaction is so central to CD4 downregulation that it has inspired exhaustive mutational
analyses (Aiken et al., 1996; Hua et al., 1997; Craig et al., 1998; Janvier et al., 2003; Lindwasser
et al., 2008; Chaudhuri et al., 2009; Mattera et al., 2011; Jin et al., 2013). These results can now be
mapped onto the structure (Figure 4). We performed additional mutagenesis to test for the functional
importance of residues that were newly identified by the structure determination (Figures 5 and 6).
Mutations in the regions of AP-2 that were already known to bind dileucine signals had the expected
loss of interaction. These include σ2 Y62A and A63D (Figure 5C). Mutations in regions unique to Nef
binding, notably σ2 R60E (Figure 5C) and α E342K (Figure 5D), also eliminated binding. Other nearby
residues with more peripheral interactions, including σ2 N48A, H85A, and C99A and α V300A, Q301A,
and N344A, had lesser mutational phenotypes, if any (Figure 5C,D). The results of these analyses are
represented in Figure 4 together with previously published data. The collective body of work and
its structural mapping are summarized in Table 2. The large majority of the mutational hits map to
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Figure 3. The AP-2 α–σ2:Nef interface. (A) Stick representation of the Nef dileucine motif (Leu164 and Leu165, orange) interacting with AP-2 σ2
(magenta), compared with a bound dileucine peptide (blue, PDB id: 2JKR) (Kelly et al., 2008). (B) Nef Glu160 of the acidic-dileucine motif forms
hydrogen bonds with AP-2 α R21 and σ2 R15. The hydrogen bond is listed as a purple dashed line. (C) Nef Glu154 in helix H4 (orange) forms hydrogen
bonds with AP-2 σ2 R10 and R61 (magenta). (D) The C-terminal part of Nef loop (171–178) interacts with both AP-2 α and σ2 (magenta). (E) The key Nef
diacidic motif Asp174 and Asp175 forms intramolecular hydrogen bonds that stabilize the loop conformation. Hydrogen bonds occur between the side
chain of Asp174 and the main-chain amide NH of Gln104, and between the side-chains of Nef Asp175 and Arg134. (F) A salt bridge between Asp108 of
Nef helix H3 bridges the Nef core to a basic patch on α.
DOI: 10.7554/eLife.01754.006
residues that directly participate in Nef-AP-2 contacts. The consistency validates both the previous
mutational approach and the structural findings.
Previous analyses had highlighted the importance of a basic patch on α comprising both Lys298-
Lys299 and Arg341. The present structure revealed that Nef Glu177 is the primary interaction partner
for α Arg341. The Nef E177K mutant manifested a reduction in both α–σ2 binding (Figure 5B) and
CD4 downregulation (Figure 6A,B), consistent with its structural role. The deepened insight obtained
from the crystal structure shows that this patch is better conceived of as a polar, rather than basic,
patch. For example, the side chain of α Glu342 has a close approach to Nef Arg178.
The finding that Nef Asp174 and Asp175 do not contact AP-2 directly was a surprise. These two
residues are required for AP-2 binding (Figure 5B) and CD4 downregulation (Lindwasser et al., 2008),
and had been expected to interact with a basic patch on α. They are, in fact, in the general vicinity of
a basic patch, close to α Lys298 and Lys299. Nevertheless, Asp174, while close to the interface, is not in
direct contact with AP-2. Its key role appears to be to anchor the turn section to the N-terminus of
helix H3. The partial positive charge at the helix N-terminus can form an interaction with Asp side-chains
that is almost as energetically favorable as a salt bridge. In this case, a short hydrogen bond is formed
with the main-chain amide group of Nef Gln104. Asp175 has a similar role in conformational stabiliza-
tion. Asp175 is partially buried in a contact with the Nef core, and forms a salt bridge with Arg134.
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Figure 4. Structural mapping of mutations that interfere with binding and CD4 downregulation. (A) The surface
representation shows the contact between AP-2 α−σ2 and Nef. (B) AP-2 α−σ2 or Nef interfaces are rotated by 90°
to expose the interaction surfaces directly to view. Interacting residues in AP-2 α are colored in yellow, residues in
AP-2 σ2 are colored in pink, and residues in HIV-1 Nef are highlighted in light blue.
DOI: 10.7554/eLife.01754.007
We hypothesized that the internal Nef Arg134-Asp175 internal salt bridge is important for stabilizing
the C-terminal turn portion of the central loop in its AP-2 binding conformation. Indeed, mutation of
Arg134 to Glu abrogated interaction of Nef with α–σ2 (Figure 5B). Moreover, co-expression in HeLa
cells of CD4 with Nef R134E followed by FACS analysis showed that this Nef mutant almost completely
lost its ability to downregulate CD4 (Figure 6A,B). These results phenocopy the D175A change
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Figure 5. Structural interface mutants of Nef and AP-2 complexes prevent binding. Y3H analysis of HIV-1 Nef and
AP-2 α–σ2 hemicomplexes with mutations of residues revealed in the crystal structure. (A) Diagram of the plasmids
used in Y3H analysis. NL4-3 Nef or mouse Tyrosinase cytosolic tail was cloned into MCS1 of pBridge and expressed
as a GAL4BD fusion protein. AP-2 σ2 or AP-1 σ1 was cloned into MCS2 of pBridge and expressed without Met.
AP-2 α or AP-3 δ was cloned into MCS of pGADT7 and expressed as a GAL4AD fusion protein. (B–D). The indicated
combinations of double transformants were plated in media lacking Leu, Trp, Met and His (−HIS),−HIS with 3-AT
(1 mM or 5 mM) or Leu, Trp and Met (+HIS). mTyr, mouse Tyrosinase cytosolic domain.
DOI: 10.7554/eLife.01754.008
(Lindwasser et al., 2008) and are consistent with a critical role for the salt bridge in stabilizing the
conformation of the Nef loop required for AP-2 binding and CD4 downregulation.
While most mutational studies of the Nef-AP-2 interaction focused on the Nef central loop, one
face of helix H3, including residues Gln104 and Asp108, contacts AP-2. The Nef mutant Q104A
behaves like wild type in both binding to α–σ2 (Figure 5B) and CD4 downregulation (Figure 6A,B).
This mutation does not alter the main-chain, and so will not affect the ability of the main-chain of
residue 104 to help anchor the central loop via Asp174. The charge-reversal mutant D108K, however,
eliminates α–σ2 binding as judged by Y3H (Figure 5B) and CD4 downregulation (Figure 6A,B). This
finding is consistent with the salt bridge seen between α Lys299 and Nef Asp108 in the structure.
Another core residue, Pro129, has limited van der Waals interactions with α, thus it was not surprising
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Figure 6. Nef interface mutants do not downregulate CD4. Nef Asp108, Arg134, and Glu177 are required for the
Nef-induced CD4 downregulation. (A) HeLa cells were cotransfected with pCMV-CD4 and pIRES-eGFP-Nef
wild-type or mutant plasmids for 24 hr. The cells were then stained with APC-conjugated anti-CD4 antibody and
Figure 6. Continued on next page
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